Stable production of a gene of interest (GOI) can be accomplished by transfecting host cells with vectors containing the GOI co-linked to a selectable marker gene, and selecting for cells harboring the GOI by inflicting a stress on the cell which can only be abated if the cell expresses sufficient quantities of the selectable marker. The vectors randomly integrate into the host cells' chromosomal DNA, and resulting transfectants show a large degree of variation in the expression level of the GOI. However, relatively few transfectant cell clones will express the GOI at the desired highest levels due to the positional effect of the site of integration. The level of expression of the genes encoded by the vector is influenced largely by the local chromosomal environment at the site of the genes' integration. (Barnett et al., 1995, Antibody Expression and Engineering, Wang and Imanaka (eds.), ACS Symposia Series, p. 604).
The use of a weakened selectable marker has been correlated with a shift towards obtaining transfectants with higher levels of expression of the linked GOI, presumably by biasing selection for integration positions that have positive influences on the expression of the weakened selectable marker and the co-linked GOI (Reff and Pfarr, 1992, Gene Amplification in Mammalian Cells, R. E. Kellems (ed.), Marcel Dekker, Inc., p.355). The expression vectors typically described in the prior art contain strong regulatory elements to drive high-level protein expression of the GOI which is co-linked to a weak selectable marker. Strategies have been utilized previously for the impairment of expression vector selectable markers, which include crippling mutations of selectable marker protein (e.g., U.S. Pat. No. 6,316,253), artificial intronic insertions in the selectable marker gene (e.g., U.S. Pat. No. 5,733,779) and impaired expression of selectable markers in polycistronic vector constructs (e.g., U.S. Pat. No. 4,713,339 and Rees et al., 1996, Biotechniques 20:102). While each of these systems for improving expression of genes of interest may have problems associated with them, it is desirable to have an improved expression system that allows for a more efficient method of generating and screening for cells that are high producers of the gene product of interest. Thus, it is desirable to create an improved expression vector and host cell expression system that can be used to efficiently generate high quantities of any recombinant protein through stable, increased expression of a gene of interest by a host cell.
The citation and/or discussion of a reference in this section and throughout the specification is provided merely to clarify the description of the present invention and is not an admission that any such reference is “prior art” to the invention described herein.